PROJECT SUMMARY/ABSTRACT While considerable progress has been made in treating primary breast cancers, metastatic breast cancers re- main a challenge. Metastatic breast cancer cells typically have chromosomal instability (CIN) that involves chromosome-level alterations leading to genomic copy number abnormalities. A major challenge in targeting breast cancers driven by CIN is the lack of known targetable alterations. We recently found that CIN pro- motes chronic inflammatory signaling in cancer cells. As chromosomes missegregate, they often become en- capsulated in micronuclei. Subsequent micronuclear rupture exposes genomic double-stranded DNA to the cytosol. Cytosolic DNA activates anti-viral innate immune pathways, chief among which is cGAS-STING signal- ing. Under normal circumstances, cGAS-STING activation promotes type I interferon and facilitates cell- mediated immunity. Engagement of STING in normal epithelial cells induces senescence and cell death. We have shown that cancer cells, however, are intrinsically resistant to cGAS-STING activation by virtue of their chronic exposure to cytosolic DNA. Instead, they upregulate alternative pathways downstream of STING, such as NF-?B signaling. The extent to which cancer cells depend on chronic inflammatory signaling is poorly un- derstood. More importantly, how they subvert innate immune signaling to avoid immune surveillance remains unknown. Our ongoing work reveals that cGAS-STING signaling is sequestered in cancer cells away from the host. Furthermore, human breast tumors upregulate ENPP1, a negative regulator of cGAS-STING signaling. ENPP1 enables immune evasion by degrading cGAMP, the second messenger produced by cGAS, only in the extracellular space. As such ENPP1 prevents host STING activation in response to tumor-to-host cGAMP transfer. Strikingly, pharmacologic inhibition of STING suppresses metastasis in syngeneic models of melano- ma, breast, and colon cancers. We postulate this is because its inhibition in tumor cells outweighs its protective role in the host. Building on this work, we will expand our pre-clinical testing of STING inhibition in breast can- cer probing its efficacy in delaying metastasis and therapeutic resistance (Aim 1). We will then examine whether cGAMP contributes toward the formation of an immune suppressive microenvironment through meta- bolic breakdown in the extracellular space (Aim 2). Finally, we will develop cGAS-STING-based biomarkers in prospectively collected tumor specimens. We will test whether the status of cGAS-STING signaling and ENPP1 levels can predict response to neoadjuvant chemotherapy and atezolizumab, an immunotherapeutic recently approved for the treatment of metastatic breast cancer (Aim 3). Our work addresses a clinically unmet need by targeting a subset of breast cancers with CIN and for which there are limited therapeutic options. If successful it will provide pre-clinical rationale for first-in-human testing of STING inhibitors for the treatment of cancer metastasis as well as the development of novel CIN-related biomarkers to predict therapeutic response.